Download Changes of diet and dominant intestinal microbes in farmland frogs

Chang et al. BMC Microbiology (2016) 16:33
DOI 10.1186/s12866-016-0660-4
RESEARCH ARTICLE
Open Access
Changes of diet and dominant intestinal
microbes in farmland frogs
Chun-Wen Chang1,2†, Bing-Hong Huang1†, Si-Min Lin1, Chia-Lung Huang1 and Pei-Chun Liao1*
Abstract
Background: Agricultural activities inevitably result in anthropogenic interference with natural habitats. The diet and
the gut microbiota of farmland wildlife can be altered due to the changes in food webs within agricultural ecosystems.
In this work, we compared the diet and intestinal microbiota of the frog Fejervarya limnocharis in natural and farmland
habitats in order to understand how custom farming affects the health of in vivo microbial ecosystems.
Results: The occurrence, abundance, and the numbers of prey categories of stomach content were significantly
different between the frogs inhabiting natural and farmland habitats. In addition, differences in the abundance, species
richness, and alpha-diversity of intestinal microbial communities were also statistically significant. The microbial
composition, and particularly the composition of dominant microbes living in intestines, indicated that the land use
practices might be one of factors affecting the gut microbial community composition. Although the first three
dominant microbial phyla Bacteroidetes, Firmicutes, and Proteobacteria found in the intestines of frogs were classified
as generalists among habitats, the most dominant gut bacterial phylum Bacteroidetes in natural environments was
replaced by the microbial phylum Firmicutes in farmland frogs. Increased intestinal microbial richness of the farmland
frogs, which is mostly contributed by numerous microbial species of Proteobacteria, Actinobacteria, Acidobacteria, and
Planctomycetes, not only reflects the possible shifts in microbial community composition through the alteration of
external ecosystem, but also indicates the higher risk of invasion by disease-related microbes.
Conclusions: This study indicates that anthropogenic activities, such as the custom farming, have not only affected the
food resources of frogs, but also influenced the health and in vivo microbial ecosystem of wildlife.
Keywords: Abundance, Custom farming, Diet, Fejervarya limnocharis, Gut microbiota, Richness
Background
The gastrointestinal tract is the primary site where microorganisms interact with the host species. The gastrointestinal microbiota maintains the functions of nutrient,
immune, and development regulation and is important
for host health [1–4]. The gut microbiota is commonly
influenced by the host diet [3, 5]. The composition of
the intestinal microbial community is suggested to result
from natural selection operating at the host level to
stabilize the gut environment and at the microbial level
to promote functional specialization [6]. The relative
abundance of symbionts and pathogenic microbes reflects the health status of the host species [7]. Microbial
* Correspondence: [email protected]
†
Equal contributors
1
Department of Life Science, National Taiwan Normal University, Taipei
11677, Taiwan
Full list of author information is available at the end of the article
interactions, e.g., resource competition, represent a deterministic factor for the dominance of the gastrointestinal microbial community [8]. Diets serve as a source of
gut microbes, which are further selected by the gastrointestinal environment.
The gastrointestinal microbes are composed of autochthonous components (residents) and allochthonous
members (hitchhikers from ingested food and waters).
Species composition of the gastrointestinal microbial
community is different from that of environmental microbes [9]. Firmicutes and Bacteroidetes are the most
abundant gastrointestinal microbial phyla [10–14] that
were suggested to descend from the early colonists in
mammalian gut evolution [6], while Proteobacteria was
found to be most dominant in tadpoles [14], house sparrow [15], and fish [16]. The codominance of these phyla
was suggested to be a consequence of niche partitioning
and metabolic complementation [6]. A healthy gut
© 2016 Chang et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Chang et al. BMC Microbiology (2016) 16:33
microbial community occupied by native bacteria could
establish a selective environment to prevent emerging
pathogens from building up a necessary population size
to cause disease. The native microbes can prevent other
similar taxa for colonization by high density blocking,
and this effect is like the “founder-takes-all” effect of the
field of population ecology [17]. However, such
“founder-takes-all” effect [17] could be vanished if fast
colonization by abundant allochthonous microbes [18].
Here, host immunity not only plays a role as a selective
pressure on intestinal microbes but also an object of natural selection by the emerging microbes.
Since the importance of relationships between the
food web complexity and species composition in an ecosystem, and between the diet content and intestinal
microbiome is emphasized in literature [19], we aimed
to explore whether the change in ecosystems as a result
of agricultural activities alters the intestinal microbial
composition of wildlife. Agricultural activities affect the
distribution of wildlife in the natural environment.
Farmland biodiversity is typically lower than that in natural fields, which is reflected in a widespread decline of
species richness and/or abundance of farmland wildlife
[20–23]. Custom farming and agricultural intensification
that rely on the use of fertilizers and pesticides recurrently create selective pressures not only on vertebrates,
but also on invertebrates [24, 25], plants [26, 27], and
soil microbes [28, 29]. The change in land use alters the
nutrient cycling of soil, which affects the diversity and
abundances of numerous environmentally important
genes of microbes [30]. Changes in nutrient cycling
affect the food webs of ecosystems [31], and changes in
heterotrophic processes and diet diversity can further
alter the digestive-tract microbiome of animals [32].
In this study, we used the rice frog Fejervarya limnocharis (Gravenhorst, 1829) (Amphibia, Anura, Ranidae) as
a system to compare the food composition (diet) and intestinal microbiota between natural and farmland frog
populations. Fejervarya limnocharis is widespread in East,
Southeast, and South Asia. In Taiwan, it is one of the
dominant amphibian species in lowland areas and is commonly found in farmlands. The Taiwanese F. limnocharis
is phylogenetically inferred as a member of the core F.
limnocharis group based on mtDNA and allozyme evidence [33, 34]. In this study, we profiled the diet and intestinal microbial composition of F. limnocharis in natural
and farmland habitats. Comparisons of the stomach contents and dominant intestinal microbes were made to
understand the changes of intestinal microbiota under the
selective pressure of environmental change, such as the
habitat variation due to agricultural activities. The multiple comparisons allowed us to address three questions:
(1) Is there any dietary difference between frogs living in
different habitats? (2) Does the intestinal bacterial
Page 2 of 13
diversity differ within and between frogs living in different
habitats? (3) Does the compositional change in intestinal
microbiota reflect the health of the in vivo microbial ecosystem in farmland frogs?
Results
Diet differentiation between habitats
A total of 26 F. limnocharis individuals (65 %, n = 40), of
which 17 originated from natural habitats and 9 from
farmlands, had stomach contents. A total of 63 individual prey items were identified to 12 orders (Table 1).
The prey consumption rate was significantly different
between the two sites (χ2 = 7.03; P = 0.008). In the natural habitat, Hymenoptera had the highest index of relative importance (IRI) score (1938.05), followed by
Orthoptera (459.68). In farmlands, the highest and the
second highest IRI scores were 2131.69 and 1310.41 in
Orthoptera and Coleoptera, respectively (Table 1). The
abundance and number of categories of stomach contents
were significantly higher in natural habitat than that in
farmlands (Z = −2, P = 0.045 and Z = −2, P = 0.036, respectively; Table 2), but the prey volume was not significantly
different between habitats (Z = −1.53, P = 0.12; Table 2).
Sequencing depth and alpha-diversity of intestinal
microbiota
The gut microbes of frog samples from the natural environment (individual labeled with N1 ~ N3) and farmland (individual labeled with F1 ~ F3) were used for
exploring the influence of land use practices on intestinal microbial composition. We generated a microbial
16S rRNA dataset consisting of 115,580 filtered high
quality, classifiable sequence reads from 133,819 raw sequence reads in total (86.37 %), with a mean number of
sequences per frog sample 19,263 ± 6868 (85.42 ±
6.83 %) (Additional file 1: Table S1). The total number
of microbial species (OTUs) of intestinal communities
characterized using a criterion of >97 % sequence similarity was 1463, with an average length of 496 bps per
sequence. The average number of OTUs of each intestinal community was 592 ± 220, ranging from 291 (sample N2) to 1011 (sample F2). The average coverage was
0.733 ± 0.061, ranging from 0.619 (sample F3) to 0.799
(sample N3). The rarefaction analysis indicated that the
sequence samplings mostly reached the plateau phase,
particularly for farmland frogs (Fig. 1). The microbial
community richness and diversity were inferred based on
the OTUs characterized using the Abundance Coveragebased Estimator (ACE) and Chao1 indices, which are nonparametric species richness estimator accounting rare
species. The ACE of the intestinal microbiota of frogs from
natural and farmland populations was 508.696 ± 114.491
and 1006.373 ± 250.663, respectively (significant difference
in t-test, t = 7.503, df = 19, p = 4.29e-07, p = 2.00e-05 under
Chang et al. BMC Microbiology (2016) 16:33
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Table 1 Stomach contents of F. limnocharis in natural habitat and farmlands
Natural habitat
Prey category
N
Farmlands
%F
%V
IRI
N
%F
%V
IRI
Insecta
Orthoptera
8
6.89
50.39
459.68
4
33.33
35.39
2131.69
Hymenoptera
27
31.03
7.36
1938.05
4
16.66
3.99
542.5
Coleoptera
3
6.89
7.21
91.83
3
25
30.99
1310.41
Blattaria
2
6.89
12.98
117.53
0
0
0
0
Hemiptera
1
3.44
0.09
7.34
0
0
0
0
Lepidoptera (Larvae)
2
6.89
0.9
34.29
0
0
0
0
Dermaptera
0
0
0
0
1
8.33
6.5
113.61
0
0
0
0
1
8.33
14.61
181.24
1
3.44
1.65
12.71
0
0
0
0
2
6.89
3.12
49.64
0
0
0
0
Chilopoda
Scolopendromorpha
Malacostraca
Isopoda
Arachnida
Araneae
Gastropoda
2
6.89
2.66
46.48
1
8.33
8.53
130.51
Oligochaeta
Stylommatophora
1
3.44
13.64
53.95
0
0
0
0
Total
49
14
N number of prey, %F percentage of frequency of each prey item, %V percentage of prey volume, IRI index of relative importance
99,999 times permutation), whereas the Chao1 index
was 522.050 ± 116.238 and 1006.272 ± 268.590, respectively
(significant difference in t-test, t = 3.972, df = 19, p = 0.0008,
p = 0.0009 under 99,999 times permutation). The intestinal
microbial community diversities in frogs from both natural
and farmland populations were significantly different in
terms of Shannon index (585.711 ± 116.074 and
1105.978 ± 289.945, respectively, t = 4.559, df = 19, p =
0.0002, p = 0.0003 under 99,999 times permutation)
but not significantly different in Simpson index
(3.977 ± 0.660 and 4.438 ± 1.030 respectively, t =
0.9947, df = 19, p = 0.3324, p = 0.3297 under 99,999
times permutation). The detailed estimates of alphadiversity are shown in Table 3.
Dominant intestinal microbial taxa in rice frogs
The abundance and richness are significantly different
between intestinal microbial species richness of F.
limnocharis from natural and farmland population. The
microbes of F. limnocharis mainly belonged to the phyla
Bacteroidetes, Firmicutes, and Proteobacteria: 35.43,
33.06, and 24.08 % in natural population, and 19.51,
46.02, and 25.22 % in farmland population, respectively
(Fig. 2b). Figure 2b showed an obvious increase of abundance in Firmicutes and decrease in Bacteroidetes in
farmland frogs compared with those in natural habitats.
The three highest-richness phyla of intestinal microbes
are Firmicutes, Bacteroidetes, and Proteobacteria (400 ±
60, 153 ± 21, and 79 ± 4 species in natural population;
511 ± 65, 207 ± 28, and 232 ± 51 species in farmland
population, respectively, Fig. 2c). Obvious increase richness in Proteobacteria was shown in farmland frogs
(Fig. 2c). We compared the identity and differences in
microbial taxa between frog samples and between different habitats and found that the farmland frogs were
composed of roughly 10 times more unique microbial
Table 2 Comparison of stomach contents of rice frog (Fejervarya limnocharis) between two habitats by Wilcoxon rank-sum test
Mean
Number of prey item
Number of prey category
Volume of prey(mm3)
Std. Dev
Median
Range
Z value
P
−2
0.045
−2
0.036
−1.53
0.12
Natural habitat
2.82
2.48
2
1 ~ 10
Farmlands
1.33
0.71
1
1~3
Natural habitat
1.76
0.9
2
1~4
Farmlands
1.11
0.33
1
1~2
140.04
71.2
2.09 ~ 396.05
26.2
32.98
0.78 ~ 78.54
Natural habitat
Farmlands
134.1
38.77
Chang et al. BMC Microbiology (2016) 16:33
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habitats. Taxa in the phyla Bacteroidetes and Firmicutes
dominated the guts of frogs from the natural population
(N1 ~ N3), whereas taxa from the phyla Proteobacteria
and Firmicutes dominated the intestines of the farmland
frogs (Fig. 4).
Classification of gut bacteria
Fig. 1 Rarefaction curves for the intestinal microbial communities of
rice frogs in natural habitats (N1 ~ N3) and farmland (F1 ~ F3) at a
difference level of 3 %
taxa than the frogs from natural habitat, particularly in
the sample F2 (Fig. 3, Additional files 2 and 3: Figure S1
and S2). In addition, there are several farmland-specific
clades in Neighbor-Joining (NJ) trees of Proteobacteria,
Actinobacteria, Acidobacteria, and Planctomycetes
(Fig. 3), indicating that the farmland environments have
created special ecological niches for these microbial
groups; in contrast, most intestinal microbes of the natural population are commonly found in farmland frogs,
indicating that these microbial species retain their ancestral traits. Most of the dominant microbes in frogs from
natural habitats were common in farmland frogs. In contrast, many dominant microorganisms in farmland frogs
were not found in frogs from natural habitats (Fig. 3,
Additional file 1: Table S2). This comparison suggests a
replacement of dominant intestinal microbes in farmland frogs. The phylogenetic analysis also showed consistent results that the main composition of intestinal
microbes of the rice frogs was different between the natural and farmland habitats. The phylogenetic grouping
of the top 10 microbes indicated an admixture of dominant microbes collected from the same habitat, but represented apparent sorting of microbes in different
Intestinal microbial organisms were classified into three
categories—generalists, specialists, and too rare—based
on the multinomial species classification method. Eight
phyla were habitat generalists (Firmicutes, Bacteroidetes,
Proteobacteria, Spirochaetae, Lentisphaerae, Deferribacteres, Cyanobacteria, and Planctomycetes), ten were
farmland specialists (Fusobacteria, Actinobacteria, Verrucomicrobia, Deinococcus-Thermus, Acidobacteria,
Elusimicrobia, Synergistetes, Chloroflexi, Gemmatimonadetes, and one unclassified phylum), one was a
natural-habitat specialist (Tenericutes), and ten phyla
were too rare to be identified (Additional file 1: Table S3
and Additional file 4: Figure S3). At the species level, we
found that 44 (8.1 %), 34 (6.3 %), 82 (15.2 %), and 381
(70.4 %) OTUs belonged to the generalist, naturalhabitat specialist, farmland specialist, and too-rare types,
respectively (Additional file 1: Table S4 and Additional
file 4: Figure S3). This classification indicated that relatively low proportions of common gastrointestinal bacteria are found in both natural and farmland habitats
and that approximately one-fifth of the bacteria differentiate the gut microbiota between habitats, while most
microorganisms are rare in these habitats. We also performed pairwise comparisons of the gut microbial community of different host individuals and found a
relatively high proportion of generalists and a lower proportion of specialists in gut bacterial communities of
frogs within habitats (generalists: 13.97 % ± 1.59 %; specialists: 26.62 % ± 2.98 %) than those seen for frogs between habitats (generalists: 9.67 % ± 0.99 %; specialists:
30.83 % ± 1.28 %). However, these differences were not
statistically significant, indicating that the individual effect cannot be neglected while explaining gut microbial
diversity (Additional file 5: Figure S4).
Table 3 Alpha-diversity of intestinal microbiota of rice frogs (Fejervarya limnocharis) at the natural habitat (N1 ~ N3) and farmland
(F1 ~ F3)
Sample
OTUs
Coverage
Community richness
ACE (95 % CI)
Community diversity
Chao1 (95 % CI)
Shannon index (95 % CI)
Simpson index (95 % CI)
N1
540
0.774
640.451 (609.752, 684.662)
653.554 (611.576, 720.152)
720.152 (4.850, 4.907)
4.907 (0.022, 0.025)
N2
291
0.753
361.315 (334.879, 403.679)
370.875 (334.723, 436.919)
436.919 (3.522, 3.583)
3.583 (0.085, 0.091)
N3
458
0.799
524.321 (501.485, 559.152)
541.720 (507.338, 600.063)
600.063 (3.398, 3.442)
3.442 (0.100, 0.104)
F1
660
0.765
797.248 (759.731, 848.877)
794.101 (749.071, 861.895)
861.895 (4.950, 5.001)
5.001 (0.018, 0.019)
F2
1011
0.687
1358.822 (1287.073, 1449.218)
1385.211 (1289.752, 1513.360)
1513.360 (5.275, 5.320)
5.320 (0.013, 0.014)
F3
590
0.619
863.048 (797.642, 949.057)
839.505 (766.514, 942.678)
942.678 (2.928, 2.993)
2.993 (0.148, 0.155)
ACE abundance coverage-based estimator
Chang et al. BMC Microbiology (2016) 16:33
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Fig. 2 Comparisons of (a) the IRI of food contents, (b) the abundance and (c) richness of intestinal microbial phyla between frogs of the natural
habitats and farmlands
Chang et al. BMC Microbiology (2016) 16:33
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Fig. 3 Microbial composition revealed in neighbor-joining trees and Venn diagram. a–f The phylogenetic analyses revealed that the microbes of
phyla Bacteroidetes and Firmicutes are mostly found in both habitats, while several microbes of phyla Proteobacteria, Acidobacteria, Planctomycetes, and
Actinobacteria are only found in farmland frogs. This result suggests that the special intestinal environment leads assemblage of unique microbes with
higher phylogenetic niche conservatism in farmland frogs. The red, blue, and green dots indicate the microbes found in frogs of natural population only,
farmland population only, and both natural and farmland populations, respectively. g Venn diagram representing the species diversity of the intestinal
microbial taxa in frog intestines. The numbers in circles represent the number of taxa. As can be seen, a higher number of unique microbial taxa were
found in farmland frogs (for which the individual F2 made the highest contribution). These results revealed that the anthropogenic interference have
altered the microbial phylogenetic distribution and species composition
Discussion
The differences in stomach contents of rice frogs may
reflect the changes in faunal species composition. There
were long-term anthropogenic disturbance such as the
use of fertilizers and pesticides in custom farming. These
anthropogenic activities may alter the faunal species
composition in farming habitat although the invertebrate
fauna of both natural and farmlands were not directly
investigated. The diet analysis showed that the food volume of rice frogs was not significantly different between
the natural and farmland habitats, but the stomach content (abundance and number of prey categories) in farmland frogs showed lower diversity than that in frogs
from natural habitats. Such dietary alteration is probably
ascribed to agricultural activities. The increase of soil life
(700 % increase of megafauna, 2500 % increase of nematodes, and 70 % increase of soil microorganisms) in 14year of monitoring conservation agriculture and organic
farming indicated the harmful effects of pesticides and
fertilizers on the terrestrial ecosystem [35]. In this study,
the decreased number of prey categories in the guts of
farmland frogs did not only point to the presence of
simplified invertebrate and plant communities in farmlands and the decreased IRI of Hymenoptera (Fig. 2a),
but also reflected the reduction of pollinator demand in
the plant community [36]. In contrast, the increase in
the IRI of Orthoptera (Fig. 2a) often suggested an increase in monocot abundance, particularly of the
Poaceae [37–39]. The dietary analysis suggested the occurrence of dramatic changes in the farmland ecosystem
due to agricultural activities.
Conventional agriculture or custom farming may
strongly alter the species composition in many ways
[35]. For example, habitat destruction during farming
may reduce diet resources and subsequently alter gastrointestinal microbiome composition in the howler monkey (Alouatta pigra) [40]. Habitat degradation following
temperature change [41] and water pollution [42] can
also subsequently alter amphibian gut microbiota. Research on the microbial composition surrounding the
forest soils revealed similar dominant microbes among
different forest types with slight difference in microbiota
[43]. This is similar to our findings that the dominant
gut bacteria are habitat generalists and most gut microbes belong to the “too-rare” type bacteria. However,
we found that approximately one-fifth of the gut bacteria
were habitat specialists (Additional file 1: Table S3 and
S4), indicating that the varied habitat indeed altered the
gut microbial composition.
Our results suggest that the estimated intestinal microbial species richness in frog guts varied within the
same order of magnitude as that in the human gut, as
estimated based on fecal analysis [10]. The gut microbiota is mainly determined by the environmental conditions where the host species reside [44]. Although
relatively few frog samples were chosen to represent the
Chang et al. BMC Microbiology (2016) 16:33
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Fig. 4 Neighbor-joining analysis for the top 10 species of intestinal microbes of rice frog in natural (N1 ~ N3) and farmland (F1 ~ F3) field
ecosystems using the 16S rRNA gene. Taxonomic hierarchies of the identified microorganisms are listed after the NJ tree. The symbol “-”
indicates unclassified
gut microbiota of rice frogs, the gut environments of different frog individuals from the same habitat may be
similar because frogs from the same habitat might share
similar niches. The digestive fluid secreted from some
specialized cells in the epithelium of the small intestine,
which maintains a stable environment in the gut [45, 46].
Therefore, the dominant gut bacteria could be similar
among frogs with similar habitats because the coexistence
of these dominant bacteria reflects the consequence of
long-term selection in the gut environment. We also
found that both dominant gut bacterial species in the natural habitat (Candidatus Hepatincola, uncultured αProteobacterium, total of 21.02 %) and farmland habitat
(Morganella sp., total of 10.95 %) were habitat specialists,
indicating that the external environment plays a major
role in the gut-dominant bacteria in frogs.
On comparing the microbial species composition, a
certain proportion of common microbial taxa (approximately 1/3 ~ 1/2) was similar between frogs from natural
habitats and farmlands, revealing the host species specificity of microbes (Additional file 3: Figure S2). The
same pattern of similar taxa in different microbial communities of diet-differentiated, geographically distant
host populations was also found in wild and laboratory
Drosophila [47] and Cylindroiulus fulviceps (Diplopoda)
with different feeding treatments [48]. However, the
higher proportion of bacterial habitat specialists than
habitat generalists suggested that external factors such
as diet, geographic variation, and the environment still
play major roles in determining the gut microbial composition (Additional file 4: Figure S3). Differences in
dominant taxa, revealed by different clusters in phylogenetic analysis (Fig. 4), suggested strong selectivity of
the microbial community in the gut environment [49].
The microbial abundances between frogs of natural
habitat and farmland are different, especially in Bacteroidetes and Firmicutes. These two phyla are the most
prevalent bacteria in digestive tracts of terrestrial animals [50, 51]. In natural population, Bacteroidetes are
the mostly abundant microbes in rice frogs’ intestines
followed by Firmicutes, but the abundance of Bacteroidetes is lower than Firmicutes in farmland population
(Fig. 2b). The Bacteroidetes of the frogs of natural habitats were mostly composed of the order Bacteroidales
(Fig. 4). Bacteroidales are known as symbiotic bacteria
essential for the digestive activity of several organisms
Chang et al. BMC Microbiology (2016) 16:33
[1, 16, 25, 48, 52, 53]. However, the abundance of Bacteroidetes decreased in farmland frogs and was replaced
by Firmicutes (Fig. 2b). Higher Firmicutes-toBacteroidetes ratio could improve the efficiency of calories uptake from food [54]. These Firmicutes microbes
were almost composed of Ruminococcaceae and Lachnospiraceae, which digest cellulose and ferment glucose
and xylose to obtain nutrients and are the prevalent bacterial families in herbivore’s digestive system [55]. The
ecological meaning of the increased abundance of Ruminococcaceae and Lachnospiraceae in farmland frogs’ intestine is unknown yet, but the high Firmicutes-toBacteroidetes ratio suggests that the alteration of food
composition might change the intestinal environments
and microbial community in farmland frogs.
The differences in dominant intestinal microbial taxa
between natural habitats and farmlands revealed in NJ
analysis indicated that the frogs acquired different bacteria due to the changes in environmental conditions. In
contrast to the high frequency of common microbial
taxa in frogs from natural habitats, the intestinal microbial communities of farmland frogs were characterized
by higher ratios of unique microbes (Additional files 2
and 3: Figure S1 and S2). These unique microbes mostly
belonged to Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes (Fig. 3). From the analysis of
the top 10 microbial organisms of every frog intestinal
samples, we found that the Classes of γ-Proteobacteria
in farmland frogs have an obvious increase in abundance
(19.75 %) than the frogs of natural habitats (0.05 %). Although γ-Proteobacteria is common in guts of diverse
taxa including amphibians [e.g., House Sparrow (Passer
domesticus) [15], leopard frog (Lithobates pipiens) [14]],
most of the γ-Proteobacteria in farmland frogs belong to
the order Enterobacteriales (72.91 %) (Fig. 4). Several intestinal bacteria that are dominant in the farmland frogs,
such as species in the genera Treponema (Spirochaetes),
Roseomonas (α-Proteobacteria), Clostridium (Firmicutes)
and genera Legionella, Acinetobacter, Pseudomonas,
Citrobacter, Morganella of γ-Proteobacteria (Fig. 4), were
probably infectious disease-related pathogens. These
bacterial genera are clinically proven to cause emerging
infectious diseases (EID) not only in humans (e.g., Legionella [56, 57]; Acinetobacter [57–59]; Pseudomonas
[60, 61]; Citrobacter [62, 63]; Morganella [64, 65]; Treponema [66, 67]; Roseomonas [68–70], and Clostridium
[71, 72]), but also in amphibian (for example, Acinetobacter would cause ulceration and necrosis in Rhinoderma darwini [73], Pseudomonas and Citrobacter
might induce immune-response in Rana pipiens [74, 75],
and so did the Morganella in Andrias davidianus [76].)
These bacteria mostly belong to the Proteobacteria. Several Proteobacteria have pathogenic or antipathogenic
functions in amphibians, for example, Janthinobacterium
Page 8 of 13
lividum in amphibian guts can inhibit the growth of lethal
amphibian fungi [77, 78]. Certain antifungal bacteria from
the genus Pseudomonas that were discovered on the skin
of amphibians, that is, the salamander (Plethodon cinereus
and Hemidactylium scutatum) [79, 80] and the frog (Rana
muscosa) [81]), were also found in leopard frogs (Rana
pipiens) [82]). The intestines of amphibians could serve as
the reservoir for these antifungal bacteria via uptake of invertebrates that have come into contact with these bacteria in the soil or by eating their own shed skin [77]. The
dynamics of these bacteria may be an indicator of host
susceptibility to these lethal fungi [83]. These Proteobacteria were previously found in smaller amounts than Bacteroidetes and Firmicutes in healthy adult mammals [19]
and human gastrointestinal samples [6] and in the frogs
from natural habitats investigated in this study.
In addition to the phylum Proteobacteria, certain bacterial genera, such as Flavobacterium in phylum Bacteroidetes and Actinobacterium and Bacillus in phylum
Firmicutes, that are chytrid-resistant or have probiotic
capabilities were mostly found in the gut of frogs in the
farmland habitat (sequence reads = 21, 59, and 4 in Flavobacterium, Actinobacterium, and Bacillus, respectively) but rarely in the natural habitat (sequence reads =
1, 4, and 0, respectively). The higher proportion of
pathogen-resistant bacteria found in farmland frogs
reflected the higher occurrence rate of harmful pathogens in the farmland habitat. The intestinal microbial
composition in frogs from farmlands was possibly under
a high risk of infectious diseases. Because stomach content is a proxy of food source in the natural environment, the differences in intestinal microbiota between
farmland and natural habitats did not only explain the
dietary alteration, but also reflected the risk of disease in
farmland wildlife due to the ecosystem alterations as a
result of anthropogenic activities.
Intestinal microbiota can possibly reflect the state of
the immune system and health of the host species [2].
Different microbial composition in frog guts may be related to pathogen resistance, for example, differential antifungal bacterium composition between populations
may be important in preventing chytrid fungus infections [84]. Differentiation of gut microbial composition
could even reflect overall body healthy of frogs. We
found lower food diversity but higher intestinal microbial richness in farmland frogs than in natural habitats.
This can possibly be explained by the recurrent selective
pressures from agricultural activities simplifying the
farmland fauna and increasing the risks of infectious disease in frog predators. Simplified food sources could also
weaken the immunity of wild animals and lead to higher
pathogen invasion [85]. Disturbance in gut microbial
composition and microbial and ecological dynamics can
lead to an increased risk of disease outbreaks in wildlife
Chang et al. BMC Microbiology (2016) 16:33
[86], or provide less anti-pathogen reservoir for preventing infection of skin pathogen [84]. Anthropogenic ecosystem alteration and pathogen or vector movements via
human or natural agencies could give rise to EID in
wildlife [87, 88]. In this study, we showed that anthropogenic interference in ecosystem (such as agricultural activities) and a change of “vectors” (food contents) can
weaken frog immunity, which is reflected in the change
of intestinal microbial climax.
Conclusions
Alterations in diet and intestinal microbial composition
in farmland F. limnocharis indicate that custom farming
influences the intestinal ecosystem of wildlife. Food may
not only play a resource role but also be one of the factors serve as a vector of infectious microbes. The intestinal microbial composition reflected the result of
intestinal environmental selection by both extrinsic (environment, such as habitat disturbance, temperature,
and food) and intrinsic (immune system) factors. In the
farmland habitat, less diverse food content and more diverse intestinal microbiota in frogs were found than in
natural habitats. This result suggests that the in vitro
ecosystem changes in vivo ecosystem. The intestinal
microbiota of F. limnocharis was determined by both environmental factors and host species, whereas the dominant intestinal microbes were more easily affected by
external environmental conditions than were the rare
microbes. The increased numbers of Proteobacteria suggested that pathogenic invasion was affected or will be
affected by the weakened immunity of farmland frogs,
which is probably caused by the heavy use of pesticides
and fertilizers in farmland. The current study revealed
the change in food resources and intestinal microbial diversity in farmland wildlife, and also suggested that outbreaks of disease-related bacteria within the gut
microbial community can reflect the damage of in vivo
and in vitro ecosystem health due to agricultural interference. It should be notified that the small sample size
of frogs in this study may not be sufficient to draw
strong conclusions, but could be indicative of changes
across habitats. Even though, this research still represents different microbial composition between habitats
and provides a reference for future studies regarding the
amphibians’ gastrointestinal microbiota.
Methods
Sampling system
Fejervarya limnocharis is a medium-sized frog with an
average body length of 4.3 cm and 5.5 cm in males and
females, respectively [89]. This species is widespread in
lowland areas at altitudes below 1500 m in Taiwan. F.
limnocharis generally aggregates around breeding ponds
during the breeding season from spring to summer. F.
Page 9 of 13
limnocharis is tolerant to human disturbance, and therefore is commonly found in farmlands. Forty frog samples
were collected from Hualin experimental forest (the natural habitat, 24°53′N, 121°33′E) and farmlands in Quchi
Community (24°55′N, 121°32′E) in Xindian (Dist., New
Taipei City, Taiwan) on September 27th, 2012. The distance between these two sites is less than 3 km. All frog
samples were brought to the laboratory of Nation
Taiwan Normal University for immediate profiling of diets and intestinal microbial composition.
Diet analyses
Frog samples were sacrificed and preserved in 70 %
ethanol. Stomach content of frogs (prey) was collected
via dissection and stored in 70 % ethanol. The length
and width of the stomach content items were measured
using cartesian papers and the prey items were identified
to order level under a stereomicroscope. The volume of
prey items was estimated using the formula proposed by
Dunham [90]. For assessing the importance of each consumed prey category, the IRI was calculated based on
the formula IRI = %O (%N + %V) [91], where %O, %N,
and %V are the percentages of the occurrence, relative
abundance, and measured volume of each prey category,
respectively, in all stomachs. The intake rate (which describes the prey eaten by F. limnocharis) between habitats was compared using the Chi-square test. The
numbers, categories, and volumes of stomach contents
were compared between habitats using Wilcoxon ranksum test. All statistical analyses were conducted using
JMP 7.0.
Intestinal microbiota
The gut microbes of six frog samples from the natural
environment (N1 ~ N3) and farmland (F1 ~ F3) were
used for exploring the influence of food composition on
intestinal microbial composition. Intestinal microbial
metagenomic DNA was extracted based on a protocol
described by Sharma et al. [92]. For each sample, we
amplified the V4 hypervariable 16S rRNA region using
the primer set 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC3′). The DNA library was constructed according to the
Roche GS FLX Titanium emPCR kit (Roche Applied Science). Pyrosequencing was carried out by Welgene Biotech Co., Ltd. (Taipei, Taiwan) using a Roche 454 FLX
titanium instrument and reagents following the manufacturer’s instructions. The V4 sequence fragments
shorter than 200 bp, without barcodes, with polyN or
polyA/T, and the reads with < Q25 were removed. Sequences with >97 % identity were treated as the same
species and as an operational taxonomic unit (OTU).
Each OTU was classified by annotating to the SILVA
database. The alpha-diversity of the gut microbiota was
Chang et al. BMC Microbiology (2016) 16:33
estimated using community richness indices, i.e., the
ACE [93], the Chao1 index [50, 94], and community diversity indices, i.e., the Shannon index and Simpson
index. Indices ACE, Chao1, Shannon index and Simpson
index between habitats were compared. Because the
small sample size could result in non-normal distribution, we regenerated 20 normalized pseudodata based on
the mean and standard deviation from the observed
data, and performed 99,999 times permutations for a
one-sample randomization test on differences of values
between habitats. Validation of t-test was given by similar p-values before and after permutations. Rarefaction
analysis was executed to measure how the gut microbial
composition of the rice frog varied depending on the
sample size.
After determination of the microbial diversity, the differences in the gut microbial composition of rice frogs
between the natural and the farmland populations was
examined by phylogenetic analysis. The neighbor-joining
(NJ) method was used for constructing a geneticdistance tree to elucidate the genetic distribution patterns of intestinal microbes between different habitats.
Nucleotide sequence alignments were performed using
the Clustal W program. The evolutionary distances were
computed using the p-distance method. The NJ tree was
constructed using the program MEGA v. 5.05 [51]. The
NJ relationships of six dominant microbial phyla Bacteroidetes, Firmicutes, Proteobacteria, Planctomycetes,
Actinobacteria, and Acidobacteria were reconstructed
separately for revealing the grouping patterns of intestinal microbes between different habitats. In addition,
every top 10 microbial OTUs of frogs (yielding a total 51
OTUs) were used to construct a genetic-distance tree
using the NJ method for clearly representing the systematic positions of the dominant intestinal microbes of
frogs in natural habitats and farmlands, with the following settings: the composite likelihood substitution model
[95], the uniform rate among sites, the heterogeneous
rates among lineages and complete deletion of gaps. A
1000 bootstrap replication was used to evaluate the supporting values for lineage grouping.
We also used the multinomial species classification
method (CLAM) [96] to classify the bacteria of generalists and specialists in two distinct habitats with the
vegan package in R. CLAM is a kind of two-group species classification method. The supermajority rule that
uses the specialization threshold value 2/3 was adopted
for determining the bacteria of habitat specialists. Under
the supermajority rule, the minimum abundance for
classification (i.e., coverage limit) was estimated and the
taxa that had abundance below the coverage limit were
considered as “too rare”. CLAM was also used for pairwise comparison between host individuals within habitats and between habitats. Comparisons of the
Page 10 of 13
proportions of generalists and specialists between
“within habitats” and “between habitats” aid in understanding the degree of individual effect on gut microbial
diversity.
Availability of data and materials
The 16S rDNA sequences identified in this study have
been deposited in the NCBI GenBank under the Bioproject PRJNA279212 (Accession number: SRX965751). Frog
samples were stored in the specimen room of Department
of Life Science, National Taiwan Normal University.
Ethics statements
Protocols of this study and the animal use were reviewed
and approved by the Ethics Committee of National Pingtung University of Science and Technology (NPUST)
(Approved No. NPUST-101-079) and the Institutional
Animal Care and Use Committee of National Taiwan
Normal University (Approved No. 101024).
Additional files
Additional file 1: Table S1. Summary of sequence reads used in this
study. Table S2. Percentages of top 10 microbial taxa of every rice frog
(Fejervarya limnocharis) sample. Table S3. List of categories of the habitat
generalists, specialists and too-rare types at the phylum level. Table S4.
List of categories of the habitat generalists, specialists and too-rare types
at the species level. (DOCX 129 kb)
Additional file 2: Figure S1. Venn diagram representing the species
diversity of the intestinal microbial taxa in frog intestines. The numbers in
circles represent the number of taxa. As can be seen, a higher number of
unique microbial taxa were found in farmland frogs (for which the
individual F2 made the highest contribution). (TIF 17137 kb)
Additional file 3: Figure S2. The Neighbor-joining tree reveals the
phylogenetic position of the intestinal microbes of Fejervarya limnocharis
in farmland only (blue), in natural habitats only (red), and in both environments (green). The phylogenetic analysis showed that roughly 1/3 clades
of microbial species are found in the farmland frogs only. (TIF 15121 kb)
Additional file 4: Figure S3. Categories of gut bacteria between
habitats classified by multinomial species classification method (CLAM).
Classification in phylum level and species level are shown. Values in plots
indicate the number of bacterial taxa (i.e. phyla and species) of each
category. F and N in the x- and y-axes indicate the farmland and natural
habitat, respectively. (TIF 156 kb)
Additional file 5: Figure S4. Pairwise comparison of gut bacterial
categories of individual hosts within habitats and between habitats. All
three comparisons of generalists, specialists, and too-rare type of bacterial
communities within habitats and between habitats are non-significant
different. (TIF 3668 kb)
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PCL conceived the study. CWC conducted the dissection experiments and
BHH conducted the molecular experiments. CWC identified the food
contents of frog samples. CWC, CLH and PCL analyzed the data. PCL wrote
the paper. CWC, BHH, SML, and PCL critically reviewed and edited the
manuscript. All authors have read and approved the final manuscript.
Chang et al. BMC Microbiology (2016) 16:33
Acknowledgements
We are grateful for the constructive comments from Chun-Yao Chen on earlier drafts of this manuscript. This research was financially supported by the
National Science Council in Taiwan (NSC 102-2621-B-003-005-MY3) and was
also subsidized by the National Taiwan Normal University (NTNU), Taiwan.
Author details
1
Department of Life Science, National Taiwan Normal University, Taipei
11677, Taiwan. 2Taiwan Forestry Research Institute, Technical Service Division,
Taipei 10066, Taiwan.
Received: 17 June 2015 Accepted: 2 March 2016
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